High-throughput technologies are generating a tremendous amount of data about cellular functions of human pathogens. However, despite the genome sequencing of hundreds of pathogens, the genomics approach has had limited success at identifying viable drug targets. One key hurdle has been that targets identified by sequencing analysis efforts do not take into account the network of interactions inside the cell; for instance, inhibiting the function of one protein may have no effect given the redundancy of pathways in the system. We propose to reconstruct and validate the metabolic and regulatory networks of Pseudomonas aeruginosa, with particular attention to previously identified mutants known to be critical for its virulence. In order to develop therapeutic strategies in the context of the intracellular networks in this human pathogen, there exists a significant need for a quantitative framework that can be used to contextualize high-throughput data, generate phenotypic predictions, and propose testable hypotheses regarding its physiology. Specifically, our proposed aims are to: (1) Reconstruct the metabolic and regulatory networks of P. aeruginosa to account for the function of 1500 genes, which will result in the largest network reconstruction of a pathogen to date; (2) Characterize the metabolic phenotypes under minimal media conditions of avirulent P. aeruginosa single-gene mutants identified previously in a signature-tagged mutagenesis screen; and (3) Analyze metabolic phenotypes of P. aeruginosa and avirulent mutant strains in cystic fibrosis-specific medium to develop possible therapeutic strategies based on conditionally essential genes. The outcome of this proposed program will be a well-characterized, well-validated model of P. aeruginosa that can be used for systematically identifying drug targets and key features of its pathogenicity, as well as a delineation of the key metabolic phenotypes (e.g., growth rate, byproduct secretions) of the STM-identified mutants that can be used potentially for the development of therapeutic strategies. The proposed program will lead to the most comprehensive reconstruction to date of a pathogen with a significant disease burden.